The ongoing epidemic of gonorrhoeae (est. 2.5 million cases per year in U.S.A.) has made Neisseria gonorrhoeae one of the most important human pathogens. The cell surface polymers of the gonococcus have been implicated as virulence factors. The gonococcus undergoes considerable phenotypic variation in these structures, the molecular basis of which is not known. In this proposal we use a combination of biochemical, genetic, and recombinant DNA techniques to increase our understanding of the mechanisms controlling surface variations in the gonococcus. Development of A More Efficient Genetic System. Since virulence genes may not have an easily selectable phenotype, in order to isolate such genes we must identify neighboring genes that do have selectable phenotypes, or screen thousands of colonies by brute force. Therefore, we plan to establish a detailed genetic map of N. gonorrhoeae. The first step is the development of efficient mechanisms of mutagensis, since the spontaneous mutation rate in the gonococcus is less than one in 10 to the nineth. Since the size of the transforming DNA is too small to span several genes, we will attempt to establish conjugation in N. gonorrhoeae. This will be accomplished by using recombinant DNA techniques to force the conjugative plasmid to integrate into the chromosome. Isolation of Specific Genes. We have developed a plasmid shuttle vector, using a chimeric N. gonorrhoeae plasmid that contains the bla gene. This will enable us to clone N. gonorrhoeae genes in E. coli and then return them to the host of origin, the gonococcus. The genes that we are most interested in cloning are those specifying virulence factors, e.g., pili, outer membrane proteins, pyocin resistance (LPS), etc. Regulation of Gene Expression. We will focus on how gene expression is controlled in the gonococcus, with attention to the mechanism of phase variation. We will sequence regions of cloned genes that are suspected to be promotors because of loss of function after random and site-specific in vitro mutagenesis. We are also attempting to elucidate the mechanisms of gene regulation in the gonococcus by studying the components necessary for in vitro transcription and translation, using N. gonorrhoeae cell-free extracts. We are currently using the gonococcal plasmids as templates in this system, and will use cloned chromosomal genes in the future.